Cytokines—Central
Factors in Alcoholic Liver Disease

Manuela
G. Neuman, Ph.D.

Manuela
G. Neuman, Ph.D., is the director of the In Vitro Toxicology Laboratory
in the Kunin–Lunenfeld Applied Research Unit, Baycrest Centre
for Geriatric Care; and an assistant professor in the Department of
Pharmacology and Institute of Drug Research, Faculty of Medicine, University
of Toronto; both positions in Toronto, Canada.

Many processes
related to the consumption or breakdown of alcohol that contribute to alcohol–induced
liver disease are mediated by small proteins known as cytokines, which are
produced and secreted by liver cells and many other cells throughout the body.
Through a variety of actions, cytokines regulate certain biochemical processes
in the cells that produce them as well as in neighboring cells. For example,
in case of an infection, they attract white blood cells to the tissues, triggering
an inflammatory response. In the liver, persistent cytokine secretion resulting
in chronic inflammation leads to conditions such as hepatitis, fibrosis, and
cirrhosis. Cytokines also regulate a process known as programmed cell death,
or apoptosis, which is in part responsible for alcohol–induced destruction
of liver tissue. Two cytokines—tumor necrosis factor alpha and transforming
growth factor beta—play prominent roles in apoptosis. Finally, a cytokine
network mediates the harmful effects of a bacterial protein called endotoxin
on the liver. Because of their diverse functions, cytokines might make attractive
targets in the prevention or treatment of alcoholic liver disease, and researchers
already have obtained encouraging results when testing such approaches. Key
words: cytokines; alcoholic liver disorder; biological activation; alcoholic
hepatitis; fibrosis; liver cirrhosis; apoptosis; tumor necrosis factor–alpha;
transforming growth factors; endotoxins; Kupffer cell; hepatocyte; disease
course; disease susceptibility; immune system; chronic AODE (alcohol and other
drug effects)

Long–term excessive
alcohol consumption can result in a spectrum of liver abnormalities, ranging
from simple fatty liver (steatosis) or fatty liver accompanied by inflammation
(steatohepatitis) to scar tissue formation (fibrosis), the destruction of
the normal liver structure (cirrhosis), and even liver cancer (hepatocellular
carcinoma). In its mildest form, fatty liver often causes no obvious clinical
symptoms and is rarely fatal. In fact, only 15 to 20 percent of chronic heavy
drinkers with steatosis have clinical liver disease, suggesting that other
factors both in the drinker’s body (e.g., genetic influences) and in
his or her environment help determine how alcoholic liver disease develops
and progresses.

For liver damage to develop,
numerous processes and biochemical reactions must occur in a variety of cells
normally located in the liver or attracted to the liver when that organ is
exposed to alcohol. This complex array of reactions is orchestrated by proteins
called cytokines, which are produced and secreted by almost all cells in the
body, including liver cells.

This article discusses
the role of cytokines in alcoholic liver disease. A review of the general
characteristics of cytokines is followed by an introduction to the process
of programmed cell death, or apoptosis, which is regulated by cytokines and
accounts for at least some of the liver damage found after chronic alcohol
consumption. Finally, the article explores how a bacterial protein called
endotoxin contributes to alcoholic liver damage by activating immune cells
in the liver to release cytokines.

Cytokines and Their Role
in Cell Communication

Almost all cells in the
body, including most types of liver cells, can produce and secrete cytokines.
Released cytokines then interact with the cells whose functions they modify
(i.e., the target cells). These target cells can be the same ones that initially
produced the cytokines; this is called an autocrine effect. In addition, the
cytokines can interact with neighboring cells; this is called a paracrine
effect. Regardless of what the target cell of a cytokine is, the interaction
occurs through a special docking molecule (i.e., a receptor) that consists
of one or more proteins. This receptor has a specific three–dimensional
shape, like a lock into which the cytokine “key” fits. The interaction
between cytokine and receptor results in subtle alterations in the receptor’s
structure, generating a communication signal that is conveyed into the cell’s
interior, where it triggers a cascade of biochemical reactions, eventually
altering the cell’s activities.

Most cytokines have more
than one effect (i.e., are pleiotropic) and can influence more than one cell
type. At the same time, many cytokines have overlapping actions. At least
in some cases, these common effects result from the fact that the receptors
for these cytokines share certain protein components and therefore mediate
the actions of multiple cytokines. For example, the receptors for six different
members of a group of cytokines called interleukins contain a protein called
the cytokine receptor gamma chain. The specific receptors for each of these
interleukins may be located on a different cell type, even within one organ;
however, because all of these receptors contain the cytokine receptor gamma
chain, their activation resulting from the binding of the various interleukins
will have similar effects on the target cells.

Although cytokines have
myriad effects throughout the body, their chief role is to help the body maintain
a steady state. By releasing and responding to cytokines, cells protect the
body against harm from foreign invaders (e.g., bacteria, viruses, and fungi)
and from damage caused by internal toxic substances (e.g., alcohol and its
breakdown products) and disease–causing cells (i.e., cancer cells).

Cytokine–Producing
Cells in the Liver

The liver consists of
several cell types that under normal circumstances produce only minimal levels
of cytokines (for a description of the various liver cell types, see the textbox).
When the liver cells—particularly immune cells called Kupffer cells—become
activated, however, cytokine production increases dramatically. If the liver
has been damaged—for example, by trauma or excessive alcohol consumption—cytokines
mediate the regeneration of liver tissue. Kupffer cells also can be activated
by the presence of disease–causing micro–organisms or substances
(i.e., pathogens). In this case, cytokines produced and released by the Kupffer
cells induce an inflammatory response in the liver (i.e., hepatitis), which
is required to start the healing process. (For a more detailed description
of the immune system and inflammatory responses, see the sidebar.) However,
if the inflammation does not subside after a short time, persistent production
of these same cytokines may lead to scar tissue formation (i.e., fibrosis)
and cirrhosis. Thus, cytokine production can have both beneficial and harmful
effects, depending on the amount and duration of cytokine release.

TEXTBOX

Liver Cell Types

The liver contains
numerous cell types, most prominently hepatocytes, endothelial cells,
Kupffer cells, and stellate cells. Hepatocytes form the bulk
(i.e., 80 percent) of the liver tissue. These cells are responsible
for breaking down (i.e., metabolizing) molecules transported by the
blood to the liver, including alcohol. Endothelial cells line
the small blood vessels (i.e., sinusoids) that distribute blood throughout
the liver. Kupffer cells belong to a group of immune cells
called macrophages. Their main function is to ingest and destroy any
foreign molecules or particles entering the liver (e.g., bacteria and
bacterial proteins). Stellate cells have two distinct functions.
When they are in a resting state, they serve to store fat in the liver.
When they become activated (e.g., as a result of a liver injury), they
produce proteins such as collagen that are required for tissue regeneration.
Excessive activation of stellate cells leads to the formation of scar
tissue, which is a characteristic of fibrosis.

END OF TEXTBOX

SIDEBAR

The Immune System
and the Inflammatory Response

The immune system,
which protects the body against potentially disease–causing micro–organisms
(pathogens) and other foreign molecules, consists of a vast array of
immune cell types whose actions and responses to foreign molecules must
be carefully orchestrated. Much of the coordination of immune system
activity is carried out by molecules called cytokines, which are produced
by certain immune cells; cytokines modify the activities of other immune
cells. In general, the immune system can be divided into two arms, both
of which involve several types of immune cells and require the actions
of various cytokines:

Innate
immunity, which responds to any pathogen it encounters. Innate
immunity exists even before the body is exposed to a pathogen for
the first time.

Acquired
immunity, which amplifies the reaction of the innate immunity.
Acquired immunity is activated only after the body is exposed to
a given pathogen for the first time, and responses of the acquired
immune system are specific to the particular pathogen. The activated
cells of the acquired immunity also constitute an immune memory—they
“remember” the pathogen to which they respond, giving
them the ability to fight a second infection by that pathogen even
faster and more efficiently.

One central component
of innate immunity is a type of white blood cell that can ingest and
thereby destroy foreign pathogens (i.e., phagocytes). Phagocytes include
cells called neutrophils, which primarily ingest invading bacteria;
natural killer cells, which eliminate cells that have been infected
by parasites or have turned into cancer cells; and monocytes, which
ingest a variety of foreign molecules and micro–organisms. Of
these groups, monocytes are of particular interest to this discussion
because they produce cytokines, which help regulate immune system activity.
In addition, monocytes display proteins called antigens (derived from
ingested pathogens and other molecules) on their surface, thereby activating
cells involved in acquired immunity to further enhance the body’s
response to the pathogen. Some monocytes stop circulating in the blood
and enter the tissues; these cells, which then are called macrophages,
have the same functions as circulating monocytes. The largest number
of macrophages resides in the liver; these macrophages are called Kupffer
cells.

The main components
of acquired immunity are white blood cells called T–lymphocytes
(T–cells) and B–lymphocytes (B–cells). T–cells
circulating in the blood and lymph recognize and bind to monocytes or
macrophages that display antigens on their surface. This interaction
activates the T–cells, causing them to multiply and produce cytokines
and chemokines. Chemokines attract additional immune cells to the site
of the infection in an effort to destroy the infected cells and thereby
contain the infection. B–cells also recognize and bind to foreign
antigens. Subsequently, B–cells begin to produce and release large
amounts of immune molecules called antibodies, which circulate throughout
the blood and bind to those antigens (or to the bacteria from which
the antigens were derived) wherever they encounter them. The antibody–covered
antigens or bacteria then can be recognized and destroyed by monocytes.

—Manuela
G. Neuman

END OF SIDEBAR

Kupffer cells, which play
an important role in inflammatory responses, as just described, are macrophages—immune
cells that enter the tissues and destroy foreign pathogens and other harmful
substances. For example, Kupffer cells eliminate bacteria and viruses that
have entered the liver, as well as damaged or abnormal liver cells, and liver
cells that have died by apoptosis (see the section “Apoptosis, Cytokines,
and Alcoholic Liver Disease”) or as the result of an inflammatory response.
Macrophages also secrete numerous cytokines that help coordinate the actions
of other immune cells in response to pathogens and toxic compounds. Macrophages
are found in all tissues, but the largest number reside in the liver.

Together with other immune
cells, macrophages such as the Kupffer cells provide the body’s first
line of defense—an acute inflammatory reaction—against bacteria
and virus infections as well as other toxic substances, such as alcohol. In
addition to ingesting and destroying pathogens, these cells secrete cytokines
that attract other immune cells to the site of the infection. The increased
blood flow that delivers those cells to the infection site, combined with
other consequences of the infection, results in the typical symptoms of inflammation—pain,
redness, and swelling. Under normal conditions, the levels of these inflammation–promoting
(i.e., pro–inflammatory) cytokines and the resulting inflammatory response
decrease once the infection is under control. If the levels of inflammatory
cytokines remain elevated, however, a chronic inflammation ensues. This is
the case in alcohol–induced chronic inflammation of the liver (i.e.,
alcoholic hepatitis), which can be a precursor to fibrosis and cirrhosis.

Based on their specific
functions, inflammation–fighting cytokines fall into the following groups
(for information on specific cytokines, see the table):

Pro–inflammatory
cytokines (e.g., interleukin[IL]–1, IL–6, tumor necrosis
factor alpha [TNF–α], and transforming growth factor beta [TGF–β]),
which stimulate the growth and development of various immune cells, activate
macrophages to release more of the same cytokines, and induce the production
of other molecules required for an inflammatory response.

Immunoregulatory
cytokines (e.g., IL–10), which help regulate the immune response
by inhibiting the proliferation of certain immune cells and promoting
the proliferation of others; reducing the production of inflammatory cytokines;
and promoting the secretion of antibodies, which bind to specific foreign
molecules, thereby inactivating those molecules and marking them for destruction
by other immune cells.

Chemokines
(e.g., IL–8), which attract a certain type of immune cell (i.e.,
neutrophils) to the site of an infection.

Cytokines Involved
in Alcoholic Liver Disease

Cytokine

Principal
Function

Pro–inflammatory
cytokines

Interleukin–1
(IL–1)

Produces inflammatory
responses; induces fever; stimulates growth and differentiation of the
immune system

Interleukin–6
(IL–6)

Promotes maturation
of antibody–secreting B cells; acts with other cytokines to stimulate
other immune system cells; stimulates production of mediators of inflammatory
responses; stimulates liver regeneration

Inhibits proliferation
of certain immune system cells and promotes proliferation of others;
reduces production of inflammatory cytokines; promotes antibody secretion

Chemokines

Interleukin–8
(IL–8)

Attracts neutrophils
to the site of an infection.

Pro–inflammatory
and immunoregulatory cytokines are discussed in more detail throughout this
article. Chemokines initially had no known biologic activity; investigators
only knew that they were associated with inflammatory diseases, such as alcoholic
hepatitis. Only after IL–8 and other chemokines called monocyte chemoattractant
protein 1 and macrophage inflammatory protein 1a and 1b all were found to
attract white blood cells to the site of an infection did it become clear
that these proteins share important structural and functional features. More
recently, studies have demonstrated that liver cells involved in tissue regeneration
(i.e., stellate cells) produce chemokine receptors and respond to the actions
of chemokines such as IL–8 (Neuman et al. 2001).

Alcohol’s
Effects on the Immune System and Cytokines

Chronic alcohol use has
adverse effects on the immune system, as shown by the fact that alcoholics
have a higher incidence of infectious diseases and deficiencies of the immune
system than nonalcoholics (Molina et al. 2002). For example, many alcoholics
are infected with the hepatitis C virus or the human immunodeficiency virus
(HIV). Both of these infections, particularly if they occur together, may
interfere with the normal networks of cytokines, possibly increasing patients’
risk of liver cancer.

As indicated above, the
liver is crucial to the body’s initial response to an infection—that
is, to the inflammatory response—because it contains large numbers of
macrophages (the Kupffer cells) that help detect toxic substances or micro–organisms
in the blood and secrete cytokines to coordinate the inflammatory response.
However, if cytokine levels remain persistently elevated in the liver (e.g.,
as a result of alcohol’s actions on the body), chronic inflammation
of the liver (i.e., hepatitis) ensues. Clinical studies have demonstrated
that patients with alcoholic liver disease have increased levels of the inflammatory
cytokines IL–1, IL–6, and TNF–α as well as the chemokine
IL–8 and other cytokines (McClain and Cohen 1989; McClain et al. 1997).
These cytokines probably are responsible for at least some of the symptoms
associated with alcoholic hepatitis, such as fever, metabolic changes, and
weight loss.

The liver also is involved
in subsequent steps of the immune response to many infections. For example,
if the primary liver cells, the hepatocytes, become infected by a virus, they
display pieces of viral proteins on their surface. These viral protein fragments
attract immune cells called T–cells, which destroy virus–infected
cells, to the liver. T–cells attach themselves to the infected hepatocytes
through the interaction between a receptor on the T–cells and the viral
protein pieces displayed together with other molecules on the surface of the
hepatocyte. This interaction leads to the destruction of the infected hepatocyte.

Chronic alcohol consumption,
however, has detrimental effects on the T–cells’ ability to destroy
the infected cells. For example, alcoholics have lower– than–normal
numbers of all types of T–cells (Szabo 1997). Moreover, alcohol may
impede the T–cells’ ability to multiply and to exert their influence
after they have been activated (Szabo 1997). As a result, the body cannot
mount an effective immune response, rendering the alcoholic more susceptible
to infections with pathogenic micro–organisms. In addition, an impaired
immune response leads to increased apoptosis of both infected and noninfected
cells, thereby contributing to liver damage.

Apoptosis, Cytokines,
and Alcoholic Liver Disease

Another way in which cytokines
contribute to alcoholic liver disease is through programmed cell death, or
apoptosis. Apoptosis is genetically determined; each cell in the body carries
the genes necessary to initiate the processes leading to apoptosis. Under
normal conditions, apoptosis helps ensure the correct functioning of the body’s
cells, as described in the following sections. Excessive or inappropriate
apoptosis, however, can lead to tissue damage, including alcoholic liver disease
(Neuman 2001).

A Brief Review
of Apoptosis

For the body to function
properly, it must ensure that damaged cells or cells that are no longer needed
can be destroyed in a safe manner. This destruction is accomplished by apoptosis.
All cells constantly survey their external and internal environments for signals
promoting cell survival or cell death. For example, the presence of growth
factors in the medium surrounding the cell indicates cell survival. Conversely,
the presence of certain other molecules in the environment or the altered
activities of certain genes in the cell are signals that promote cell death.
Every cell monitors and integrates the sometimes–conflicting signals
it receives to decide whether to live or commit suicide. When the “death
signals” prevail, the cell initiates a series of biochemical reactions
that result in its death.

To avoid putting additional
stress on the organism, apoptosis proceeds so that no inflammatory reaction
is initiated (see figure 1). When a cell undergoes apoptosis, it severs its
contacts with neighboring cells and begins to form small protrusions (i.e.,
blebs). Next, the cell’s nucleus breaks apart and the DNA breaks into
small pieces. These nuclear and DNA pieces, as well as other cell components
(i.e., organelles) are distributed among the blebs, which increase in size.
Each bleb eventually encloses a portion of the cell’s content, and the
cell breaks apart, forming several so–called apoptotic bodies which
can then be ingested and destroyed by macrophages and other cells. In this
manner, none of the cell content is released to cause an inflammatory response.

Figure 1
Schematic representation of the process of apoptosis.

In the liver, apoptosis
may play an important role in eliminating hepatocytes that no longer are needed
or whose DNA has been damaged. For example, under certain conditions the number
of liver cells increases. When those excess cells are no longer needed, apoptosis
probably helps decrease liver mass again. In general, hepatocytes seem to
be particularly susceptible to apoptosis (Neuman 2001).

Several endogenous compounds
such as cytokines, or foreign compounds such as alcohol and other drugs, can
trigger hepatocyte apoptosis. A better understanding of the role that apoptosis
plays in alcohol–induced liver diseases and of the mechanisms by which
apoptosis occurs in the liver may give researchers insights into these diseases
and point to possible treatments.

Alcohol–Induced
Apoptosis

Numerous factors related
to alcohol and its breakdown, which occurs primarily in the liver, can induce
apoptosis of hepatocytes. As described in other articles throughout this journal
issue, the processes by which alcohol is broken down in the hepatocytes generate
a variety of molecules that can be toxic to the liver or interfere with normal
physiological processes (see figure 2). For example, alcohol breakdown through
the enzyme known as cytochrome P450 2E1 (CYP2E1) leads to the formation of
small oxygen–containing molecules called reactive oxygen species (ROS).
These ROS, unless they are rapidly eliminated or converted into harmless molecules
by antioxidants, can interact with and damage complex molecules in the cells
(e.g., proteins and DNA). (For more information on ROS and their harmful effects,
see the article in this issue by Wu and Cederbaum.) Both increases and decreases
in the levels of ROS can lead to apoptosis of hepatocytes.

Figure 2
Pathways through which alcohol (ethanol) can contribute to apoptosis.
Alcohol is broken down (i.e., metabolized) in the liver cells by two
enzymes, alcohol dehydrogenase (ALD) and, particularly after chronic
alcohol consumption, cytochrome P450 2E1 (CYP2E1). Both enzymes convert
alcohol to acetaldehyde, a toxic substance. Some of the acetaldehyde
interacts with proteins in the cells, forming compounds called adducts
that can activate certain immune cells to produce various cytokines,
including interleukins (ILs), interferon gamma (IFN–γ), and
tumor necrosis factor alpha (TNF–α). In addition to acetaldehyde,
alcohol metabolism by CYP2E1 also generates highly reactive molecules
known as reactive oxygen species (ROS), which accumulate primarily in
cell structures called mitochondria. ROS normally are eliminated from
the cells by compounds known as antioxidants, particularly a small molecule
called glutathione (GSH). Alcohol, however, depletes the cell’s
GSH stores, thereby further exacerbating ROS accumulation in the mitochondria.
This process leads to the release of cytochrome c from the
mitochondria, which then activates enzymes called caspases and promotes
production of IL–8 in the cell. Finally, alcohol leads to increased
levels of a bacterial protein called endotoxin in the blood and in the
liver, which activates immune cells called Kupffer cells that reside
in the liver. These cells then produce TNF–α, which in turn
activates another type of liver cell, the stellate cells, to produce
transforming growth factor beta (TGF–β) and collagen, a protein
involved in scar tissue formation (fibrosis). TNF–α production
also leads to increased production of chemokines (e.g., IL–8),
which attract inflammatory cells from the bloodstream to the liver,
contributing to liver inflammation. Excess TNF–α and chemokine
production also causes increased production of adhesion molecules that
play an important role in fibrosis. Thus, all of these diverse pathways
contribute to inflammatory reactions and fibrosis and culminate in the
induction of apoptosis and organ damage.

Alcohol and its breakdown
not only result in the formation of ROS and other reactive molecules in the
cell but also reduce the levels of certain antioxidants. Decreases in an important
antioxidant, glutathione (GSH), in the cells have been shown to be an early
event in apoptosis. GSH is found both in the fluid filling the cells (i.e.,
the cytosol) and in small cell components called mitochondria, in which most
of the cell’s energy production occurs. For the cell to function normally,
it is important that enough GSH is present in the mitochondria because most
of the ROS are formed there. Unless these ROS are rapidly eliminated by GSH,
they can damage the mitochondria, allowing the molecule cytochrome c
to leak from the mitochondria into the cytosol. Once in the cytosol, cytochrome
c can activate enzymes known as caspases that can trigger apoptosis.

Alcohol can reduce GSH
levels in the cells, particularly in the mitochondria. For example, when isolated
hepatocytes were exposed to alcohol, GSH levels in the mitochondria (but not
in the cytosol) decreased dramatically (Neuman et al. 1998).1 (1
For reviews of the metabolism of GSH, see Meister and Anderson 1983; Meister
1988.) Hirano and colleagues (1992) showed that GSH depletion is partly responsible
for cytotoxicity caused by alcohol. Additional experimental studies have suggested
that the depletion of GSH in the mitochondria could be a result of impaired
GSH transport from the cytosol (Garcia–Ruiz et al. 1995a,b;
Fernandez–Checa et al. 1996; Kaplowitz et al. 1996; Kaplowitz and Tsukamoto
1996; Neuman et al. 2002).

GSH depletion may be one
of the factors contributing to alcohol–induced apoptosis. In addition,
alcohol exposure led to the activation of caspase 3 and other signals triggering
apoptosis. When alcohol–exposed hepatocytes were viewed under an electron
microscope, damage to the mitochondria was the first indication that the cells
were undergoing apoptosis (Katz et al. 2001). These hepatotoxic effects of
alcohol and its breakdown products are amplified by responses from the immune
system (e.g., activation of T–cells and release
of pro–inflammatory cytokines).

Another element linked
to hepatocyte apoptosis is a system consisting of two molecules, Fas and Fas
ligand. Fas, also known as CD95, is a type of receptor found on hepatocytes
and is similar to one of the receptors for the cytokine TNF–α (also
discussed in the next section). This Fas receptor can interact with the Fas
ligand, which is present on the surface of certain T–cells. The interaction
between the Fas ligand and soluble Fas triggers chemical processes in the
hepatocyte that lead to apoptosis. As a result of these processes, apoptosis–inducing
caspases are activated, the mitochondria become leaky, and other apoptosis–promoting
reactions are initiated. One of the factors that may promote binding of Fas
and the Fas ligand is the presence of ROS—for example, those ROS generated
by the breakdown of alcohol (Navder and Lieber 2002). Thus, ROS can adversely
affect hepatocytes by damaging the cell’s DNA, promoting apoptosis induced
by the Fas/Fas ligand system, and altering the activities of various other
genes in the cells.

Tumor Necrosis
Factor Alpha (TNF–α). TNF–α is one of
the inflammatory cytokines; among other functions it triggers the production
of additional cytokines. In the liver, these cytokines together attract inflammatory
cells to the organ, destroy hepatocytes, and initiate a healing response that
includes the formation of scar tissue. The structure of TNF–α is
similar to that of the Fas ligand; consequently, TNF–α shares some
of the functions of Fas ligand, such as promoting apoptosis. For example,
researchers have found that TNF–α can induce apoptosis in cells
derived from a human liver tumor (i.e., hepatoma cells) (Neuman et al. 1998)
as well as in hepatocytes obtained from mice and rats (Leist et al. 1997;
Kurose et al. 1997a,b). TNF–α also may play a role in
alcohol–induced apoptosis. For example, studies have found that whereas
the liver (and other tissues) normally produces only minimal levels of TNF–α,
the levels of that cytokine were substantially increased in the blood of alcoholic
patients with acute or chronic liver disease (Pastorino and Hoek 2000).

The ultimate effect of
TNF–a on hepatocytes in an animal or human is strongly influenced by
the presence of other cytokines in liver tissue. In normal mice, TNF–α
is essential for liver regeneration. If part of an animal’s liver is
removed, TNF–α assists in liver regeneration by promoting the proliferation
of hepatocytes. If the mice lack the pro–inflammatory cytokine IL–6,
however, increased production of TNF–α resulting from partial removal
of the liver promotes the death of hepatocytes (Tilg and Diehl 2000). Similarly,
destruction of the gene for the regulatory cytokine IL–10 (which helps
terminate inflammatory responses) exacerbates TNF–mediated liver injury
in mice (Tilg and Diehl 2000). Conversely, mice that are deficient in several
other cytokines—such as IL–18 and interferon–gamma, which
promote TNF–α activation and, consequently, apoptosis—are
protected against the apoptosis–promoting effects of TNF–α
(Tilg and Diehl 2000).

Consequences
of Alcohol Exposure on the Effects of TNF–α. Studies
have found that alcohol may increase the liver’s sensitivity to inflammatory
cytokines, such as TNF–α, in two ways. First, alcohol may directly
or indirectly stimulate Kupffer cells to produce and release TNF–α
into the small channels (i.e., sinusoids) in which the blood flows through
the liver. (One indirect mechanism—the alcohol–induced increase
in the levels of a bacterial endotoxin in the blood—is discussed in
the next section.) Second, alcohol may enhance the sensitivity of hepatocytes
to TNF–α (Nanji et al. 2001). TNF–α might increase the
metabolism of the hepatocytes, particularly energy production in their mitochondria,
which could lead to an increased production of ROS in the mitochondria. These
ROS, in turn, could activate a regulatory protein called nuclear factor kappa
B (NFκB), which controls the activities of numerous genes, including
those that encode TNF–α and one of its receptors, as well as genes
encoding proteins that promote apoptosis. Thus, a vicious cycle would be established
in the hepatocytes: TNF–α promotes ROS production, ROS production
activates NFκB, and NFκB leads to enhanced production of additional
TNF–α and its receptor as well as to production of factors that
promote apoptosis. This cycle eventually alters the structure of the hepatocytes,
impairs their function, and can lead to hepatocyte apoptosis.

Transforming
Growth Factor Beta (TGF–β). The second cytokine involved
in regulating apoptosis is TGF–β, generally considered an immunoregulatory
cytokine. It is similar in structure and function to a large group of molecules
that activate or inhibit various growth and differentiation processes. TGF–β
has a range of effects, including triggering apoptosis in a variety of normal
and tumor cells. For example, TGF–β, together with TNF–α,
was reported to induce apoptosis in human hepatoma cells (Katz et al. 2001).

Consequences
of Alcohol Exposure on the Effects of TGF–β. Studies
have found that more TGF–β is produced in the livers of patients
with alcoholic cirrhosis than in the livers of healthy people, suggesting
that TGF–β might be involved in the development of alcohol–induced
liver damage (Neuman et al. 2002). Moreover, this cytokine can cause the hepatocytes
to produce more of certain molecules (i.e., transglutaminase and cytokeratins)
that normally are responsible for giving the cells their shapes. When excess
levels of these molecules are present, however, they become cross–linked
to form microscopic structures called Mallory bodies, which are indicators
(i.e., markers) of alcoholic hepatitis (Cameron and Neuman 1999).

TGF–β also
can contribute to liver damage by activating a type of liver cell called stellate
cells. In a resting state, these cells primarily serve to store fat and vitamin
A in the liver. When activated, however, stellate cells produce collagen,
the major component of scar tissue. Collagen production by activated stellate
cells is a crucial step in the development of fibrosis in patients with alcoholic
steatohepatitis. Therefore, TGF–β may be an important player in
this process.

As with TNF–α,
hepatocytes normally produce little or no TGF–β. Under certain
conditions, however, hepatocytes can produce TGF–β or take up the
cytokine from the outside (Bedossa and Paradis 1995). Alcohol might trigger
the activation of TGF–β and thereby contribute to the initiation
of apoptosis if this molecule enters the blood in higher concentrations (Neuman
2001; Katz et al. 2001).

Bacterial Endotoxin,
Cytokines, and Alcoholic Liver Disease

One of the factors that
can enhance the production of TNF–α in macrophages, including Kupffer
cells, thereby promoting apoptosis, is a bacterial protein called endotoxin
or lipopolysaccharide (LPS). Endotoxin is released from the bacteria normally
living in the intestine when those bacteria die. If endotoxin enters the blood
and reaches the liver, it interacts with Kupffer cells, activating the cells
to produce cytokines. (For more information on endotoxin and its effects,
see the article in this issue by Wheeler.) Endotoxin–stimulated cytokine
production may play a crucial role in the onset of liver failure during sepsis2
in alcoholic patients (Keshavarzian et al. 1999; Mathurin et al. 2000; Jerrells
et al. 1998). (2 Sepsis is an infection or contamination [e.g.,
of a wound].)

In a healthy person, endotoxin
is mostly confined in the intestine and does not reach the bloodstream and
the liver. Alcohol consumption, however, can lead to increased endotoxin levels
in the blood by altering the permeability of the intestinal wall, allowing
endotoxin to cross that wall more easily. This phenomenon, also known as “leaky
gut,” was demonstrated in animal studies. In these studies, direct administration
of endotoxin into the intestine led to increased endotoxin levels in the blood
entering the liver in animals that had received an alcohol–containing
diet, but not in animals that had not received alcohol (Neuman et al. 2002).
Other animal studies found that the “leaky gut” phenomenon also
occurred after a single alcohol dose and was more pronounced in animals already
suffering from alcohol–induced liver injury (Mathurin et al. 2000).
Researchers do not yet know, however, whether the prevalence and severity
of increased gut permeability is higher in heavy drinkers with cirrhosis or
severe liver injury than in heavy drinkers without alcoholic liver disease.

In the liver, endotoxin
interacts primarily with Kupffer cells, and this interaction is considered
crucial to the secretion of TNF–α, which then interacts with receptors
on both Kupffer cells and hepatocytes, leading to the production of other
inflammatory cytokines, such as IL–1, IL–6, and IL–8. The
importance of this chain of events to the development or progression of alcoholic
liver disease in humans was demonstrated by several observations:

The concentrations
in the blood of TNF–α and the other inflammatory cytokines
were increased in patients with alcoholic hepatitis (McClain and Cohen
1989).

Patients with the
highest cytokine concentrations in the blood had the most severe disease,
as indicated by the highest rate of in–hospital mortality (McClain
and Cohen 1989).

Concentrations in
the blood of both TNF–α and TNF receptors were correlated with
the levels of endotoxin in the blood and with the stage of liver disease
in patients with alcoholic liver disease.

Taken together, these
findings indicate how important intestinally derived endotoxin and endotoxin–induced
cytokines, such as TNF–α, are to the development of alcoholic hepatitis.
This conclusion was further confirmed by animal experiments in which the activity
of TNF–α was inhibited in different ways. For example, the animals
were treated with antibodies that inactivate TNF–α, or the experiments
were conducted in animals that lack the receptor for TNF–α (Molina
et al. 2002). In both conditions, the animals were protected from alcohol–induced
liver disease, even after long–term alcohol exposure.

Researchers have investigated
the mechanisms through which endotoxin leads to TNF–α production
using a mouse model in which alcohol was directly administered into the animals’
stomachs. This approach was used to compare the effects of alcohol administration
in normal mice and in mice genetically engineered to lack the receptor with
which endotoxin interacts on the Kupffer cells—a molecule called CD14.
The study found that although alcohol administration caused liver injury in
the normal mice, it had only minimal effects in the CD14–deficient mice
(Yin et al. 2001). In addition, the CD14–deficient animals showed no
induction of TNF–α and no activation of NFκB (which is regulated
by TNF–α). These findings confirm that the binding of endotoxin
to its receptors on the Kupffer cells and the resulting Kupffer cell activation
and production of TNF–α are key events in the development of alcoholic
liver injury. This idea is further supported by results from a recent study
in human alcoholic patients, which demonstrated that a specific variant of
the CD14 gene is associated with an increased risk of alcoholic liver injury
(Jarvelainen et al. 2001).

Analyses in rodents also
have suggested that females may be more susceptible to alcohol–induced
increases in endotoxin levels in the blood than males. In one study, researchers
assessed liver damage and measured the levels of the mRNAs—intermediate
molecules that are formed when the genetic information encoded in a gene is
“read” to produce the gene’s product—for TNF–α
and other cytokines in male and female rats receiving an alcohol–containing
diet. The investigators found that female rats exhibited more severe alcohol–induced
liver injury than male animals (Nanji et al. 2001). In addition, the female
rats had higher endotoxin levels in the blood as well as higher levels of
several cytokines, including TNF–α. Finally, the female animals
showed greater NFκB activation. These findings suggest that compared
with males, increased endotoxin levels in the blood of females stimulate NFκB
activation and cytokine production, thereby enhancing liver injury.

In summary, endotoxin
seems to be an important contributor to the development of early alcoholic
liver disease. In recent years, researchers have made important progress in
understanding the mechanisms underlying endotoxin’s effects as well
as the consequences of these effects in alcoholic liver injury. Nevertheless,
several key questions remain unanswered. For example, investigators do not
yet know whether endotoxin participates in advanced alcoholic liver disease
and what mechanisms may underlie such an effect. Answers to these questions
could provide the scientific rationale for developing and evaluating new therapeutic
approaches aimed at inhibiting endotoxin’s effects in heavy drinkers
with severe liver injury.

Conclusions and Future
Perspectives

Many factors acting in
concert contribute to the sensitivity of liver cells to alcohol exposure and
the resulting liver damage. As described in this article, cytokines have pivotal
functions in many of these processes, such as the activation of the immune
system that leads to chronic liver inflammation, initiation of hepatocyte
apoptosis, and the effects of endotoxin. Accordingly, researchers and clinicians
are trying to determine the clinical benefits that might flow from research
on cytokines in the field of alcohol–induced liver disease. Some of
the investigations will focus on the exact factors determining a person’s
susceptibility to alcoholic liver disease. Only 20 percent of heavy drinkers
actually develop liver disease (Molina et al. 2002), suggesting that some
heritable factors might be involved in determining whether liver damage occurs.
Other investigations should address the possibility that susceptibility to
liver disease is the result of an “innate immune reactivity” to
alcohol. Alternatively, in people with high susceptibility to alcoholic liver
disease, liver cells may fail to sense alcohol–induced damage to neighboring
cells and the resulting apoptosis–promoting signals or to remove apoptotic
cells or apoptotic bodies from the liver. All of these possibilities require
further investigation.

Because they have such
a wide range of effects, cytokines are important targets in the management
of alcoholic hepatitis and other inflammatory conditions. Once researchers
fully understand how cytokines work, they may be able to develop clinical
uses for cytokines and their receptors as well as for antibodies that can
inhibit cytokine activity. Researchers also can generate antibodies against
molecules normally found in the body (e.g., cytokines). If administered to
a person or animal, these antibodies will interact with their target, thereby
interfering with the activities of that molecule. For example, antibodies
that interact with TNF–α may be able to inhibit the cytokine’s
harmful actions, and the effectiveness and safety of these antibodies in treating
patients should be evaluated. Other cytokines, such as the immunoregulatory
cytokine IL–10, already are being tested in clinical trials of patients
with alcoholic hepatitis. The observation that stellate cells, which are essential
for the repair of damaged liver tissue (but also for the development of fibrosis),
respond to the actions of the chemokine IL–8 also may lead to clinical
applications.

As researchers have elucidated
many of the processes occurring during apoptosis and the factors that contribute
to it, they also have identified many potential points for regulating these
processes that might be useful in treating alcoholic liver disease. For example,
it may be possible to regulate factors influencing the induction of apoptosis
or to enhance the elimination of apoptotic cells. Future research in apoptosis
may provide new treatment options and increase our knowledge of the pathogenesis
of human liver diseases, including alcohol–induced liver damage.